1. Field of the Invention
The present invention relates generally to the field of genetic molecular tools, particularly those molecular tools that may directly or indirectly direct translation and the activity of ribosomes. The invention also relates to the field of pharmacologically active molecule screening methods, as genetic molecular tools are provided that are used in a variety of screening methods.
2. Related Art
Co-translational protein folding and maturation are often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has at least two important limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components are different from concentrations in vivo.
Newly synthesized polypeptide chains first pass through the ribosomal exit tunnel, which spans ˜100 Å between the peptidyl transferase site and the surface of the ribosome1,2 (FIG. 1A). The tunnel protects 30-40 aa of the nascent chain from contact with other cellular components, and restricts the accessible conformational space3,4. As the nascent chain lengthens, its N-terminus emerges into the cytosol. At this point, the chain has access to additional conformational space, and may also interact with other cellular components.
The rate of nascent chain synthesis (˜20 aa/sec in E. coli) is considerably slower than many folding events, some of which occur on a microsecond timescale. The difference between these rates implies that protein folding can begin during chain synthesis, and co-translational folding has been detected experimentally5-9. Co-translational folding therefore represents a fundamentally different starting ensemble for folding than dilution of a full length chain out of a chemical denaturant10, and conformations populated by nascent chains in vivo can be populated quite differently (or not at all) during refolding in vitro6,9. For a given protein, co-translational folding might therefore modify the dominant folding pathway, potentially influencing aggregation propensity. Yet, while recent studies have made some progress in tracking and observing the folding of proteins within intact cells11-13, the earliest steps of folding in vivo (while chain synthesis is underway) remain unclear, in part because of a lack of tractable methods to dissect these early folding steps.
Translation mixtures (and intact cells) include ribosomes bearing nascent chains of all lengths. This heterogeneity means there currently are no biophysical techniques available to assess nascent chain conformation as translation occurs. Uncoupling chain elongation and folding, typically by increasing the population of ribosomes bearing nascent chains of a discrete length, can, however, provide ‘snapshots’ of nascent chain conformations during synthesis.
Producing stalled ribosomes bearing nascent chains of a uniform length remains a substantial technical hurdle for measuring ribosome-bound nascent chain conformations. Current methods to create stalled E. coli ribosome-nascent chain complexes are technically quite challenging, particularly for longer nascent chain lengths.
The above and other limitations in the art associated with ribosome-nascent chain manipulation currently limit the efficiency and potential powerful applications for using ribosomes in various biophysical analytical techniques, including ribosome display. A need continues to exist in the art for more reliable and reproducible techniques for capturing and monitoring translational events involved in the genesis of proteins, as well as protein conformation events related thereto.